Mycotoxins in Crude Building Materials from Water-Damaged Buildings

We analyzed 79 bulk samples of moldy interior finishes from Finnish buildings with moisture problems for 17 mycotoxins, as well as for fungi that could be isolated using one medium and one set of growth conditions. We found the aflatoxin precursor, sterigmatocystin, in 24% of the samples and trichothecenes in 19% of the samples. Trichothecenes found included satratoxin G or H in five samples; diacetoxyscirpenol in five samples; and 3-acetyl-deoxynivalenol, deoxynivalenol, verrucarol, or T-2-tetraol in an additional five samples. Citrinine was found in three samples. Aspergillus versicolor was present in most sterigmatocystin-containing samples, and Stachybotrys spp. were present in the samples where satratoxins were found. In many cases, however, the presence of fungi thought to produce the mycotoxins was not correlated with the presence of the expected compounds. However, when mycotoxins were found, some toxigenic fungi usually were present, even if the species originally responsible for producing the mycotoxin was not isolated. We conclude that the identification and enumeration of fungal species present in bulk materials are important to verify the severity of mold damage but that chemical analyses are necessary if the goal is to establish the presence of mycotoxins in moldy materials.     

The androgen receptor CAG repeat length polymorphism associates with prostate volume in Finnish men with benign prostatic hyperplasia

The development of benign prostatic hyperplasia requires the presence of testicular androgens during prostate development, puberty, and ageing. We thus examined the association of three polymorphisms, namely, CYP3A5 6986A>G, CYP19A1 1531C>T, and androgen receptor (AR) gene CAG repeat length, which have previously been linked to the androgen pathway and with clinical characteristics of benign prostatic hyperplasia. Tissue samples from 262 consecutive prostate operations were used for genotyping. Prostate volumes and prostate-specific antigen values were collected from patient records. Linear regression analysis was performed to study the polymorphisms in an age-adjusted model. We did not find any association between the CYP3A5 6986A>G polymorphism and clinical characteristics of benign prostatic hyperplasia. Further, the previously published CYP19A1 1531C>T polymorphism association with an enlarged prostate could not be confirmed with this material. However, we detected an association between short AR gene CAG repeat length and a small prostate volume, which confirms a previous finding in the Finnish population. The data presented suggest a negligible role for the CYP3A5 6986A>G polymorphism in benign prostate enlargement in the Finnish population. However, the results presented do provide further evidence for potentially different genetic mechanisms behind benign prostatic hyperplasia in Finnish and other Caucasian populations. This is based on the conflicting results for AR gene CAG repeat length associations with benign prostatic hyperplasia found in published works.     

Patterns of divergence among conifer ESTs and polymorphism in Pinus sylvestris identify putative selective sweeps

Finding genes that are under positive selection is a difficult task, especially in non-model organisms. Here, we have analyzed expressed sequence tag (EST) data from 4 species (Pinus pinaster, Pinus taeda, Picea glauca, and Pseudotsuga menziesii) to investigate selection patterns during their evolution and to identify genes likely to be under positive selection. To confirm selection, population samples of these genes have been sequenced in Pinus sylvestris, a species that was not included in the EST data set. The estimates of branch-specific Ka/Ks (nonsynonymous/synonymous substitution rates) across all genes in the EST data set were similar or smaller than estimates from other higher plant species. There was no evidence for the traditional indication of positive selection, Ka/Ks above 1. However, several lines of evidence based on polymorphism patterns suggest that genes with high Ka/Ks (0.20-0.52) in the EST data set are in fact more affected by positive selection in P. sylvestris than genes with low Ka/Ks (0.01-0.04). The high Ka/Ks genes have a lower level of polymorphism and more negative Tajima's D than the low Ka/Ks genes. Further, in the high Ka/Ks group, the Hudson-Kreitman-Aguade test is significant. This suggests that the EST data set is a good starting point for finding genes under positive selection in conifers and that even moderate Ka/Ks values could be indicative of selection. A group of 5 genes with high Ka/Ks collectively show evidence for positive selection within P. sylvestris.     

Colonization routes of Pinus sylvestris inferred from distribution of mitochondrial DNA variation

Understanding the present-day distribution of molecular variation requires knowledge about the history of the species. Past colonization routes and locations of refugia of Scots pine (Pinus sylvestris) were inferred from variation in mitochondrial DNA in material collected from 37 populations located in countries within, and immediately adjacent to the continent of Europe. Two mitochondrial regions, nad1 intron (exon B/C) and nad7 intron 1, were included in the study. Differentiation in maternally inherited mitochondria was high (G _ST′ = 0.824). Two new haplotypes were found at the nad7 intron 1. The occurrence of a 5-bp indel variant was restricted to the Turkish Kalabak population and a 32 bp only found in Central, Eastern, and Northern Europe. The complete absence of the 32-bp indel from the Mediterranean peninsulas supports the view that coniferous forests existed outside these areas during the last glacial maximum, and these populations contributed to the subsequent colonization of the northern parts of Europe. P. sylvestris shares features of its glacial and postglacial history with two other northern, cold-tolerant tree species, Picea abies and Betula sp. These three species differ from many other European trees for which pollen core and molecular evidence indicate colonization from southern refugia after the last glacial period.     

Genetic variability in natural populations of Arabidopsis thaliana in northern Europe

Ten populations of the model plant Arabidopsis thaliana were collected along a north-south gradient in Norway and screened for microsatellite polymorphisms in 25 loci and variability in quantitative traits. Overall, the average levels of genetic diversity were found to be relatively high in these populations, compared to previously published surveys of within population variability. Six of the populations were polymorphic at microsatellite loci, resulting in an overall proportion of polymorphic loci of 18%, and a relatively high gene diversity for a selfing species (HE = 0.06). Of the overall variability, 12% was found within populations. Two of six polymorphic populations contained heterozygous individuals. Both FST and phylogenetic analyses showed no correlation between geographical and genetic distances. Haplotypic diversity patterns suggested postglacial colonization of Scandinavia from a number of different sources. Heritable variation was observed for many of the studied quantitative traits, with all populations showing variability in at least some traits, even populations with no microsatellite variability. There was a positive association between variability in quantitative traits and microsatellites within populations. Several quantitative traits exhibited QST values significantly less than FST, suggesting that selection may be acting to retard differentiation for these traits.     

Assembly of stable human type I and III collagen molecules from hydroxylated recombinant chains in the yeast Pichia pastoris. Effect of an engineered C-terminal oligomerization domain foldon

The C-propeptides of the pro alpha chains of type I and type III procollagens are believed to be essential for correct chain recognition and chain assembly in these molecules. We studied here whether the 30-kDa C-propeptides of the human pC alpha 1(I), pC alpha 2(I), and pC alpha 1(III) chains, i.e. pro alpha chains lacking their N-propeptides, can be replaced by foldon, a 29-amino acid sequence normally located at the C terminus of the polypeptide chains in the bacteriophage T4 fibritin. The alpha foldon chains were expressed in Pichia pastoris cells that also expressed the two types of subunit of human prolyl 4-hydroxylase; the foldon domain was subsequently removed by pepsin treatment, which also digests non-triple helical collagen chains, whereas triple helical collagen molecules are resistant to it. The foldon domain was found to be very effective in chain assembly, as expression of the alpha 1(I)foldon or alpha 1(III)foldon chains gave about 2.5-3-fold the amount of pepsin-resistant type I or type III collagen homotrimers relative to those obtained using the authentic C-propeptides. In contrast, expression of chains with no oligomerization domain led to very low levels of pepsin-resistant molecules. Expression of alpha 2(I)foldon chains gave no pepsin-resistant molecules at all, indicating that in addition to control at the level of the C-propeptide other restrictions at the level of the collagen domain exist that prevent the formation of stable [alpha 2(I)]3 molecules. Co-expression of alpha 1(I)foldon and alpha 2(I)foldon chains led to an efficient assembly of heterotrimeric molecules, their amounts being about 2-fold those obtained with the authentic C-propeptides and the alpha 1(I) to alpha 2(I) ratio being 1.91 +/- 0.31 (S.D.). As the foldon sequence contains no information for chain recognition, our data indicate that chain assembly is influenced not only by the C-terminal oligomerization domain but also by determinants present in the alpha chain domains.     

DNA sequence variation in BpMADS2 gene in two populations of Betula pendula

The PISTILLATA (PI) homologue, BpMADS2, was isolated from silver birch (Betula pendula Roth) and used to study nucleotide polymorphism. Two regions (together about 2450 bp) comprising mainly untranslated sequences were sequenced from 10 individuals from each of two populations in Finland. The nucleotide polymorphism was low in the BpMADS2 locus, especially in the coding region. The synonymous site overall nucleotide diversity (pis) was 0.0043 and the nonsynonymous nucleotide diversity (pia) was only 0.000052. For the whole region, the pi values for the two populations were 0.0039 and 0.0045, and for the coding regions, the pi values were only 0 and 0.00066 (for the corresponding coding regions of Arabidopsis thaliana PI world-wide pi was 0.0021). Estimates of pi or theta did not differ significantly between the two populations, and the two populations were not diverged from each other. Two classes of BpMADS2 alleles were present in both populations, suggesting that this gene exhibits allelic dimorphism. In addition to the nucleotide site variation, two microsatellites were also associated within the haplotypes. This allelic dimorphism might be the result of postglacial re-colonization partly from northwestern, partly from southeastern/eastern refugia. The sequence comparison detected five recombination events in the regions studied. The large number of microsatellites in all of the three introns studied suggests that BpMADS2 is a hotspot for microsatellite formation.     

Exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus

Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptor-mediated endocytosis, escapes from endosomal vesicles to the cytosol, and then replicates in the nucleus. We examined the role of the microtubule (MT)-mediated cytoplasmic trafficking of viral particles toward the nucleus. Immunofluorescence and immunoelectron microscopy showed that capsids were transported through the cytoplasm into the nucleus after cytoplasmic microinjection but that in the presence of MT-depolymerizing agents, viral capsids were unable to reach the nucleus. The nuclear accumulation of capsids was also reduced by microinjection of an anti-dynein antibody. Moreover, electron microscopy and light microscopy experiments demonstrated that viral capsids associate with tubulin and dynein in vitro. Coprecipitation studies indicated that viral capsids interact with dynein. When the cytoplasmic transport process was studied in living cells by microinjecting fluorescently labeled capsids into the cytoplasm of cells containing fluorescent tubulin, capsids were found in close contact with MTs. These results suggest that intact MTs and the motor protein dynein are required for the cytoplasmic transport of CPV capsids and contribute to the accumulation of the capsid in the nucleus.     

Hypothermia preserves myocardial function and mitochondrial protein gene expression during hypoxia

Hypothermia before and/or during no-flow ischemia promotes cardiac functional recovery and maintains mRNA expression for stress proteins and mitochondrial membrane proteins (MMP) during reperfusion. Adaptation and protection may occur through cold-induced change in anaerobic metabolism. Accordingly, the principal objective of this study was to test the hypothesis that hypothermia preserves myocardial function during hypoxia and reoxygenation. Hypoxic conditions in these experiments were created by reducing O2 concentration in perfusate, thereby maintaining or elevating coronary flow (CF). Isolated Langendorff-perfused rabbit hearts were subjected to perfusate (Po2 = 38 mmHg) with glucose (11.5 mM) and perfusion pressure (90 mmHg). The control (C) group was at 37 degrees C for 30 min before and 45 min during hypoxia, whereas the hypothermia (H) group was at 29.5 degrees C for 30 min before and 45 min during hypoxia. Reoxygenation occurred at 37 degrees C for 45 min for both groups. CF increased during hypoxia. The H group markedly improved functional recovery during reoxygenation, including left ventricular developed pressure (DP), the product of DP and heart rate, dP/dtmax, and O2 consumption (MVo2) (P < 0.05 vs. control). MVo2 decreased during hypothermia. Lactate and CO2 gradients across the coronary bed were the same in C and H groups during hypoxia, implying similar anaerobic metabolic rates. Hypothermia preserved MMP betaF1-ATPase mRNA levels but did not alter adenine nucleotide translocator-1 or heat shock protein-70 mRNA levels. In conclusion, hypothermia preserves cardiac function after hypoxia in the hypoxic high-CF model. Thus hypothermic protection does not occur exclusively through cold-induced alterations in anaerobic metabolism.